Chromatography and capillary electrophoretic methods are two means used frequently for the separation of biopolymers. Because of the limitation of the quantity of the employed mobile phase in capillary electrophoretic method, however, such methods can generally only be used on an analytical scale. Chromatography is now the most important and the most effective separation method for the separation, renaturation and purification of biopolymers. It would be desirable to be able to use such technique not only for analytical scale, but also for production scale.
When chromatography is used for separation, it is commonly accepted that the separating effect is proportional to the number of column plates, e.g., column length. If the column is too long, however, it is too expensive and it leads to a higher column pressure, which must be controlled with a high performance liquid chromatograph. It is reported in the literature that evidence can be obtained showing effective biopolymer separation with a short column using the stoichiometric displacement model for solute-in-liquid chromatography; and, a packed column of 2 mm in length using a slice of membrane having a thickness of about 2 mm that is cut off from a continuous rod is used to make biopolymer separations with good results when used on an analytical scale. It is still unknown, however, whether such a short column with stable effects can be utilized for industrialized production, as well as to what degree such a column can be shortened and still be effective.
At present, a column for high performance liquid chromatography employed at a production scale level is generally packed with particles having a diameter of about 20–30 μm. It is ideal that the ratio of column length to diameter be about 10 or less to avoid a too-high column pressure under conditions of high flow rate, thereby causing decreasing separation effects. In order to ameliorate the fact that the volume of the column bed always becomes effectively smaller under the pressure accumulated by the soft matrix over an increased column length, Pharmacia Co., which is famous around the world for its production of chronographic media adapted for use at low and middle pressure conditions, has offered a cake-shaped chromatographic column with shorter thickness (but having a length of at least 10 cm) and of greater diameter. When in use, several cake-shaped chromatographic columns of this type would typically be connected together in series. Therefore, the sum of the lengths of these serially connected columns is still many times greater than the diameter of an individual cake-shaped column, so the flow rate through this series of chromatographic cakes still cannot be too high when it is applied.
Many proteins expressed with E. coli in biotechnology exist in the form of inclusion body in E. coli because of its high hydrophobicity. Although the primary structures of such proteins may be correct, their third or fourth structures are basically wrong. As the inclusion body generally has the property of high hydrophobicity, it should be dissolved with a denaturant at high concentration, such as 7.0 mol/L guanidine hydrochloride (GuHCl) or 8.0 mol/L urea. Then the renaturation of the proteins can be processed. In the current technology, the dialysis method and the dilution method are commonly used for protein renaturation. Nevertheless, these two methods not only have a low renaturation effect (on the order of about 5%–20% generally), but they also require too much time resulting in failure to realize the objective of separating impure proteins. One alternative technology has been developed in which a denatured protein is renatured and simultaneously purified by high performance hydrophobic interaction chromatography (HPHIC). However, if some precipitates of proteins occur by sample injection, the chromatographic column in this technique will be blocked or damaged. Thus, the denatured facilities currently used for protein renaturation and purification and the multifunctional protein-purifying unit as described above have major limitations which reduce their utility and effectiveness.
It can be concluded that the following major problems currently exist in the separation and purification of proteins in various biotechnology processes:
1. Separation and purification of biopolymers made in a glass column, a plastic column or a stainless steel column, packed by soft based media, and having large diameters. The shortcomings of these processes include low column efficiency, need for large volumes of media, high consumption of mobile phase, and long production periods.
2. When small solutes are separated with a chromatographic column, the resolution should be proportional to the column length. The ratio of column diameter to column length is normally about 1:10. The column length has a small effect on the resolution of biopolymers. In general, a column with a length of about 5 cm is selected. When such a column is packed with small particles, the chromatographic system through pressure is obviously increased. Such a column should therefore be used with a high-pressure liquid chromatograph, but this results in increased production costs. Such increased costs counterbalance some of the advantages of using a column packed by small particles, namely high efficiency, large volume and good reproducibility.
3. For the usual chromatographic columns, it is preferred that samples having a high viscosity not enter the columns, and that no more than a small quantity of precipitates from samples are allowed to settle in the column head. In practice, however, the inlet and outlet conditions of the mobile phase cannot readily be changed, so such control is not convenient for the operation.
4. In a usual separation and purification process, relatively pure products can be obtained only through the sequential steps of renaturation, removal of denaturants, coarse purification, and multistep fine purification. These steps represent a long and cumbersome processing technology usually involving great loss of mass and bioactivity and, thus, with a relatively low recovery (generally no higher than about 5–20%).
5. Common renaturation methods include a dilution method and a dialysis method. With the dilution method, many dilution steps should be taken to gradually decrease the concentration of the denaturing agent employed. Thus, such a process brings many handling difficulties for the later separation and purification steps if, at each stage, samples are diluted tens, or even hundreds, of times. The dialysis method, on the other hand, takes too much time (e.g., 24 hours for only one dialysis step generally), and, furthermore, the dialysis agent should be changed many times. In addition, with the above two renaturation methods, the subject proteins are easily aggregated and precipitated resulting in a long renaturation time during the renaturation process.
6. There are many steps involved in the current separation and purification methods. In the course of such separation and processing steps, the volume of solution containing the subject proteins is always increasing. Also, each step of these methods requires substantial associated processing equipment. Therefore, current separation and purification techniques require a relatively large investment in equipment with correspondingly high production costs.
At present, for packing and manufacturing a chromatographic column, satisfactory column efficiency can be obtained when chromatographic packing material is packed using familiar axial and radial pressurization techniques. But these two methods are only applicable to the packing process for a relatively long chromatographic column wherein the ratio R of column length to diameter is greater than unity (i.e., R>1). There are no ideal packing and manufacturing methods currently available, however, for the process of packing a chromatographic “cake” wherein the ratio R of cake thickness to cake diameter is smaller than or equal to unity (i.e., R≦1). If the traditional technology were used to pack a chromatographic cake wherein R≦1, the following problems would likely occur:
1. In the traditional process to pack a chromatographic column, liquid goes through the column inlet and column outlet in the axial direction (i.e., along the axis of the column). In general, the traditional packing processes are only applicable to a chromatographic column wherein the ratio R of column length to column diameter is greater than unity.
2. Because the technique of packing columns properly requires a high level of skill, if an operator is lacking the necessary skills, circumstances may occur in which a column is not packed properly, for example having a tight outlet and/or a loose inlet. An improperly packed column has low reproducibility of results because of the defect in the column packing.
3. In a conventional chromatographic column packing process, the inlet end of the column can be made even and smooth with a blade after the column has been packed. For a chromatographic cake, however, there are no easy and effective methods to make such a large surface area of the packing, such as the inlet end of the column, even and smooth after packing the cake.
4. In general, a chromatographic column needs to be repacked after long use at high pressure. Before a column is repacked, the sunken inlet end of a chromatographic column is typically repaired to prolong column life. In order for the sunken inlet end of a chromatographic column to be repaired, however, the column head must be dismantled to remove the frit. For a column with a relatively small diameter, it is generally easy to remove the frit because of the small surface area of the frit. But, the surface area of the inlet end of a “chromatographic cake” is generally more than one up to 100 times or more greater than that of a comparable conventional chromatographic column. Also because the distributor is tabled tightly with the frit, and is normally also relatively tightly pressed in the groove of the column body, it is difficult to remove it. If it is removed with force, the distributor could often be damaged.
5. When packing having a small diameter is to be packed into a chromatographic cake using the high pressure slurry method, possible leakage must be tested before the cake can be packed because the diameter of a chromatographic cake is relatively great and, accordingly, it is difficult to seal it off if there is a leak. After the chromatographic packing is packed, if there is still a leakage problem, it can create great difficulties for repairing the apparatus.
6. For a relatively large chromatographic cake, a relatively large slurry tank and associated devices are required which increase the production costs.